In the last few years, great advances have been made in the development of chips (microelectronic devices) capable to characterise and identify analytes of interest. In general, a chip or multisensor for the detection of analytes is composed of a substrate over which is placed a plurality of individually addressable analysis sites (IAASs). Each IAAS includes a selectively immobilised specific receptor. This receptor can be of biological nature, for instance, antibody, enzyme, oligonucleotide, etc., or a living biological system, for example, cell, tissue, live organism, etc., or of chemical nature, for example, aptamer, imprinted polymer, zeolite, etc., which recognises the analyte that selectively binds to or interacts with that receptor. When a solution that contains a sample with one or several analytes is put in contact with a chip that has one or more binding sites that have modified IAASs with specific receptors for those analytes, a receptor-analyte interaction is produced on the corresponding IAASs and, consequently, the presence of those analytes can be deduced in the assay sample and their concentration can be quantified by appropriate transduction schemes.
Selective deposition of molecules on the IAASs is very important in industrial scale manufacturing of arrays (devices that have a repetitive microelectronic architecture) for genetic analysis, chemical sensors, enzymatic and affinity biosensors and, microinterfaces for direct communication between microelectronic devices and living beings (like, for example, the direct control by the nervous system of biomechanical implants).
One of the methods followed by the industry to manufacture oligonucleotide arrays includes the application of site-addressable techniques based on photolithographic activation. This photolithographic activation uses solid-phase chemical synthesis (in situ), that is compatible with fabrication techniques employed in the semiconductor industry. Using a series of photolithographic masks to define chip exposure sites, followed by specific chemical synthesis steps, the process constructs high-density arrays of oligonucleotides, with each probe in a predefined position in the array. The resolution of this technique is of the order of 1 μm or even less (0.1 μm). It is ideally limited by the wavelength of radiation used for patterning of the array. The disadvantage of this technique is that there is no possibility for the control of quality of the synthesised probes and, consequently, a redundancy in the number of hybridisation sites is necessary to assure reliability.
Other approaches to produce biochips (arrays in which the immobilized molecule is biological) use different microfluidic contact and non-contact printing technologies, which allow dispensing volumes of liquids in the range of the nanoliter. These contact and non-contact printing methods have the advantage that the biochemical compounds can be preselected in compliance with quality control standards. However, the resolution of the method (density of points) is one or two orders of magnitude smaller than the photolitographic activation.
Electrochemical methods are cheaper than the above-mentioned strategies. These methods include the electrochemical copolimerization of pyrroles and modified oligonucleotides with pyrrole groups. Ideally, the resolution of these techniques is also limited by the photolitographic limit of the design of the array. Besides, these techniques also allow a previous selection of oligonucleotides based on their quality. Consequently, they are more advantageous than the techniques previously mentioned, but, maybe, require more time for their design and might present the drawback of non-selective deposition. In addition, liquid chemistry is used for the production, which is not a standard in the semiconductor industry.
Colloidal gold is adequate for the immobilisation of mercapto-modified molecules, which form dative bonds with the gold surface. The use of colloidal gold is known to immobilise enzymes in sensors through electrodeposition. Crumbliss and collaborators [Crumbliss et al. (1992), Biotechnology and Bioengineering, 40:483–490], combined glucose oxidase (GOx), peroxidase (HRP) and xanthine oxidase (XO) with colloidal gold and electrodeposited those conjugates in platinum or vitreous carbon applying +1,6V (vs. Ag/AgCl saturated) for 2 hours. These enzymatic electrodes gave an electrochemical response to the corresponding enzymatic substrates in presence of mediators of the ferrocene family. Through this study they demonstrated the utility of the colloidal gold as vehicle of biocompatible deposition appropriate for the elaboration of enzymatic electrodes. Yabuki and Mizutani [Yabuki S. and Mizutani F. (1995), Denki Kagaku, 63(7): 654–659] also conjugated GOx with colloidal gold and deposited the conjugate on vitreous carbon, gold and platinum, by means of the same process, and observed that the intensity currents were ten times smaller using platinum or gold than with vitreous carbon, probably due to the lesser quantity of adsorbed conjugate in the metallic electrodes. However, none of these previous articles mentions the possibility of selective deposition with micrometric or sub-micrometric resolution.